Altered glycosylation of glycolipids, glycoproteins, or proteoglycans are frequently observed in tumor tissues and cultured cancer cell lines. These changes have diagnostic use but also can contribute to the ability of tumor cells to grow, induce neovascularization, metastasize, and avoid host immune surveillance. We are primarily studying sulfated and sialylated glycoconjugates and their interaction with extracellular matrix and cell surface adhesion molecules, including thrombospondins. We previous demonstrated that heparan sulfate proteoglycans mediate inhibitory activities of thrombospondin-1 for T cell activation and proliferation as well as upstream modulation of Ras and MAP kinase signaling. We are isolating the proteoglycans that mediate these activities from T cells and examining the specificity of thrombospondin-1 for binding to proteoglycans required for signaling by angiogenic growth factors. Using metabolic labeling, we have demonstrated that T cells express several proteoglycans and release them into the medium. A 200 kDa secreted proteoglycan was identified that binds to TSP1. Inhibitors of these interactions are being tested for effects on angiogenesis and tumor growth.Although TSP1 clearly affects functional responses of mature immune cells, its role in development of the immune system has not been studied. We compared the cellular composition of various lymphoid organs in adult wild type and TSP1 null mice and found that maturation of T cell subsets appeared to be normal in thymus of TSP1 mutant mice. Expression of the activation markers CD25 and CD69 did not differ from those of wild type mice. However, all thymocyte subsets from the TSP1-deficient mice exhibited consistent and significant reductions in cell surface level of CD44 relative to the same subsets from wild type mice. In the spleen, percentages of B cells, NK cells, macrophages, CD4+ and CD8+ T cells were also the same in TSP1 mutant and wild type mice, but all splenocyte subsets in TSP1 null mice displayed significantly decreased levels of CD44. CD44 mediates cell-cell and cell-matrix interaction in a large part through its affinity for the polysaccharide hyaluronan. TSP1 is not a direct ligand for CD44 or hyaluronan, but both interact with a number of other proteins. Some of these may also be TSP1 ligands. Future experiments will further characterize the mechanism for dysregulation of CD44 in TSP1 transgenic mice and the functional consequences of altered CD44 expression in the physiological functions of TSP1 and the immune pathology of TSP1 null mice.Because TSG-6 modulates CD44-mediated cellular interactions with hyaluronan, we examined the possibility that TSP1 interacts with TSG-6. We found that recombinant full length human TSG-6 (TSG-6Q) and Link module of TSG-6 (Link_TSG6) bind 125I-TSP1 with comparable affinities. Trimeric recombinant constructs containing the N-modules of TSP1 or TSP2 inhibit binding of TSP1 to TSG-6Q and Link_TSG6, but other recombinant regions of TSP1 do not. Therefore, the N-modules of both TSP1 and TSP2 specifically recognize the Link module of TSG-6. Heparin, which binds to these domains of both proteins, strongly inhibits binding of TSP1 to Link_TSG6 and TSG-6Q, but hyaluronan does not. Inhibition by heparin is due to its binding to TSP1, because heparin also inhibits TSP1 binding to Link_TSG6 mutants deficient in heparin binding. Removal of bound Ca2+ from TSP1 reduces its binding to full-length TSG-6. Binding of TSP1 to Link_TSG6, however, is enhanced by chelating divalent cations. In contrast, divalent cations do not influence binding of the N-terminal region of TSP1 to TSG-6Q. This implies that divalent cation-dependence is due to conformational effects of Ca-binding to the C-terminal domains of TSP1.